HPLC analysis for comparison between β glucanstandard (up) and sample β -glucan (down) 

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... which were marked using artistic software on the photos. Serum levels of VEGF were quantitatively determined in mouse serum and control subjects by means of quantitative sandwich enzyme linked immunosorbent assay ELISA test using ready kit (R and D Systems, USA). A group of Swiss albino male BALB/c mice (8- 10 week old, 20-25 g in weight) were used throughout this study. The animals were housed in plastic cages and maintained in hygienic conditions at temperature around 25° C and were fed with suitable quantity of water and complete diet. Seven groups of mice were used in this experiment and treated as follows: Group I: Control (3 mice): treated with 0.1 ml of phosphate buffer saline. Group II: glucan treatment (3 mice), treated with 0.1 ml of β -glucan (250 μ g/ml). Group III: glucan treatment (3 mice), treated with 0.1 ml of β -glucan (500 μ g/ml). Group IV: glucan treatment (3 mice), treated with 0.1 ml of β -glucann (750 μ g/ml). Group V: glucan treatment (3 mice), treated with 0.1 ml of β -glucan (1000 μ g/ml). Group VI: glucan treatment (3 mice), treated with 0.1 ml of β -glucan (1500 μ g/ml). The phosphate buffer saline and β -glucan were administrated intraperitoneally for ten successive days, and the mice were sacrificed by the end of the tenth day. Blood samples were taken and allowed to clot for 2hrs at room temperature before centrifuged at 1,500 rpm for 20 min. Serums were aspirated and kept at -20° C until use. A one way analysis of variance ANOVA (Duncan) was performed to test whether group variance was significant or not. Data were expressed as mean ± standard error and statistical significances were carried out according to program statistical package for social sciences IBM SPSS, version 20. The total dry weight of β -glucan obtained was 8.8% of dry yeast cells. Glucan can be extracted from backer’s yeast with varying degrees of purity depending on the method used; the chemical, physical properties and biological activity which may vary according to the method of extraction (Courie et al., 2005). Zechner-Krpan et al. (2010), showed that the yield of β -glucan from yeasts using alkaline – acid extraction was up to 12%. Many procedures used for yeast’s β -glucanextraction were based on alkaline – acid applications with differences in application time, the type and the concentrations of the chemicals. The advantage of using alkaline-acid extraction method was the treatment with base, acid and organic solvent which leads to dissolve or remove most the proteins, mannan, nucleic acids and other components (Al-Rubaee, 2008). The β -glucan may contain high percentage of impurities which may directly affect its physical and chemical properties that causes loss the ability to absorbin water and this was the basic feature for using the β -glucan in the food industry (James et al., 1991). For this reason more chemical analyses were preformed to estimate the purity of the extracted β -glucan. Chemical analyses of the β -glucan extracted from Saccharomyces cerevisae HS-1 were performed first by estimating the carbohydrate and protein contents. Results indicated that the percentage of carbohydrates and proteins of the extracted β -glucan were 44% and 0.45% respectively. The components of both glucose and protein gave an important indication about β -glucan purity; therefore the high amount of sugars with few percent of proteins indicating that the method of choice was good in yielding β -glucan with very small amount of impurities. The glucan purity is an important character in determination of its application since glucan may be used in pharmaceutical, cosmetics, food and other felids (Vetvickova, 2007). Lee et al., (2001), reported the ability to yield 32% glucanwith 0.8% proteins by using alkaline–acid extraction method. Moreover, FT-IR spectroscopy was used to detect the functional group in the chemical structure of β -glucan, and compared these groups with standard groups. Result in Fig. (1A) showed that infrared -1 spectrum at the absorbance 1041.5 cm means the presence of C-O-C bonds which is a characteristic feature for β -glucan structure -1 stretching with the standard 1051 cm (Fig.1B) (Hozova et al., 2007). The absorbance at -1 (1384.8 cm ) refers to the presence of C-H aliphatic bending; the standard absorbance -1 was at 1375 cm (Karreman et al., 2006). On the other hand free hydroxyl groups and carboxyl groups were absorbed at regions -1 -1 2862.2 cm and 2923 cm that found in the carbohydrate (Ibrahim et al., 2006).In addition, the extracted β -glucan had appearance typical to that of the standard β -glucan with high degree of purity and absence of the protein contents that absorbed at 1635, 1542, 1650 -1 cm (McCann et al., 2007). On the other hand, the technique was used to determine the quality and purity of S. cerevisiae β -glucan, in addition to confirm the structural similarity with the standard β -glucan. The HPLC analysis revealed one major peak 3.78 of a liquid sample β -glucan (Fig. 2), which indicating the purity of the extracted β -glucan. Such peak showed the same retention time of the β -glucan standard indicating the efficient method of the extraction. The anti-angiogenic activity of the S. cerevisiae β -glucan was detected by CAM assay. Table (1), revealed the result of this assay. According to the results, the concentration 250 μg/egg of β -glucan had a slight non-significant inhibition effect on neovascularization as compared with the negative control and the number of the blood branches was 58±1.45, Fig. (3b). On the other hand, the concentrations (500, 750 and 1000 μg/egg) revealed a significant inhibition of the neovascularization process with a decrease in blood vessel branches 53, 50 and 43, respectively (Fig. 3 c, d, e).It was noticed that the increases in the concentration of β -glucan, will inversely decrease the chick CAM angiogenesis and by reaching the concentration of 1500 μg/egg, 50% of CAM capillarizations were significantly inhibited (Fig. 3 f). Results indicated that β -glucan extracted from S. cerevisiae affects the generation of the new blood vessels and hence reduces the number of blood vessels branches. The decrease of the neovascularization may be attributed to several lines of evidence that might support the role of polysaccharides ( β -glucan) in anti- angiogenisis process. First, β -glucan is most effective in promoting the secretion of IL-12 that has been shown to directly inhibit angiogenesis of endothelial cells (Del Vecchio et al., 2007), β -glucan injection intraperitoneally to the mice caused a decreases in VEGF level and leads to the activation of the MNC to secret IL-12 (Chin et al., 2005). Second, the VEGF is a factor that regulates the formation of blood vessels, injection the mice with concentration (500, 750, 1000, 1500 μg/egg) of β -glucan deceased VEGF and inhibited the neovascularization (Del Vecchio et al., 2007). Many studies utilized the β glucan of fungi as anti-angiogenic factor, with different molecular weights of different medical fungi (Chin and Seviour, 2007). Liu et al. (2013) showed that the polysaccharide from the Antrodia cinnamomea confirmed its anti- angiogeniceffect. The effect of blood phagocytic cells against yeasts cells was examined with the presence of the extracted β -glucan. Result in the Table (2) indicates that the coefficient values of the phagocytosis increased with increasing β -glucan concentrations with a significant effect of p ≤ 0.05. The highest rate of the phagocytosis coefficient reached up to70% at the concentration 1500 μg/ml, while there is no effect at the concentration 250μg/ml. However, with concentrations 500, 750, 1000 μg/ml the effect was significantly higher than the control and 250 μg/ml but no differences in effect recorded between these concentrations. It's clear that β -glucan has the ability to activate the phagocytic cells and increase their ability to engulf the dead yeast cells when the blood phagocytic cells interact with β -glucan. The phagocytosis is one of the mechanisms of the body defense against the pathogen; monocyte, macrophage and polymorphic nucleus cells are the main cells in this respect and they represent as the first line of the body defense, they found in most of the body tissues (Vetvicka and Vetvickova, 2008). β -glucanis one of the immune modulators especially that extracted from microorganisms. The immune modulators have the ability to activate the immune cells in order to pass in the states of activation leads to production of some compounds that play important roles in immune organization like (IL-12 and TNF α ) in addition to inflammatory mediators which increase the ability of the immune cells to defense against pathogens (Vetvicka and Vetvickova, 2009). β -glucans is recognized as pathogen- associated molecular patterns (PAMPs) by several mammalian immune cell receptors, such as dectin-1, toll-like receptors, complement receptor 3(CR3). These receptors allow the β -glucan to interact with immune cells, such as macrophage, neutrophils and lymphocyte; such interactions will activate several intracellular pathways responsible for the immune pharmacological properties (Medeiros et al., 2012). Macrophage is one of the immune cells that recognize the β -glucan and play critical role in all phases of the host defenses that are both innate and adaptive immune responses, the secretions of (IL-12 and TNF α ) and inflammatory mediators are affected by the macrophage cells activation, therefore the activation of these cells by β -glucan increases host immune defense (Akramiene et al., 2007). Al-Rubaee, (2008), indicated that increasing in phagocytosis activity for the polymorphic nuclear cells (PMNc) up to 74% when yeast beta glucan was used as modulator. Al-Aubydi and Abed (2011), depended on β -glucan as immunomodulator and indicated that the β -glucan have a positive effects in animals immune system. The coefficient value of the VEGF concentration decreased ...